The long-term objective of the proposed research is to apply transplantation of germ line stem cells as a new approach to generate germ line transgenic large animal models of human and animal disease. Introduction of genetic modifications through the male germ line will shorten the time necessary to produce transgenic animal models and provide a strategy that is potentially more efficient compared to the current method to induce transgenesis in non-rodent animals by somatic cell nuclear transfer. The testis stem cell is unique among the stem cells in an adult male in that it is the only cell type that divides and can contribute genes to subsequent generations, making it an ideal target for genetic modifications. The pig is used as a nonrodent model species because of its importance for biomedical research. Due to the size and physiology, results obtained in pigs are expected to be applicable to other large animal species and man. The aims of this revised renewal project are 1) to improve transgene transmission through germ cell transplantation, 2) to provide an efficient system for in vitro propagation of porcine germ line stem cells, and to generate a transgenic pig model through manipulation of the male germ line;3) to explore alternate sources of donor cells for modification and transplantation;and 4) to explore xenografting of isolated testis cells from pigs and nonhuman primates as accessible models for the study and manipulation of spermatogenesis in large animals and man. Experiments will explore lentiviral and non-viral germ cell transduction systems, quantify effects of germ cell depletion on efficiency of donor-derived spermatogenesis in recipient pigs, investigate novel strategies of germ cell culture, including transient stimulation of germ cell proliferation using an SV40 Large T antigen-VP22 fusion protein system, and induction of germ cell differentiation in pluripotent cells. Generation of a transgenic pig model expressing a fluorescent marker in pluripotent cells and in the germ line will provide 'proof of principle'that the strategy developed in this project serves as an efficient alternative to cloning for the generation of transgenic large animal models. In addition, this pig model will provide a valuable resource for future research aimed at derivation of pluripotent porcine stem cells and for germ line differentiation from stem cells. De novo morphogenesis of functional testis tissue after grafting of isolated cells to mouse hosts will be further developed as a complementary approach to germ cell transplantation to the testes of homologous recipient animals. This novel approach allows reconstitution of spermatogenesis from isolated cells in an in vivo culture system, a process that is currently not possible in vitro. This system will serve as a bioassay for germ line stem cell potential and will enable screening of genetic changes targeted to specific testicular cell types prior to reconstitution of spermatogenesis. Overall, the novel, complementary strategies of homologous germ cell transplantation and testis cell grafting to be further developed in this proposal will enable efficient germ line modification in a large animal model.